1 Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing 2 Li Zhang1*, Ngoc-Tung Tran1*, Hairui Su1*, Rui Wang2, Yuheng Lu11, Haiping Tang4, Sayura Aoyagi10, 3 Ailan Guo10, Alireza Khodadadi-Jamayran1, Dewang Zhou1, Kun Qian5, Todd Hricik3, Jocelyn Côté6, 4 Xiaosi Han8, Wenping Zhou7, Suparna Laha9, Omar Abdel-Wahab3, Ross L. Levine3, Glen Raffel9, 5 Yanyan Liu7, Dongquan Chen12, Haitao Li4, Tim Townes1, Hengbin Wang1, Haiteng Deng4, Y. George 6 Zheng5, Christina Leslie11, Minkui Luo2, and Xinyang Zhao1 7 8 1. Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, The University of 9 Alabama at Birmingham, Birmingham, AL 35294, USA. 10 2. Program of Molecular Pharmacology, Sloan Kettering Institute, New York, NY 10021, USA. 11 3. HOPP, Sloan Kettering Institute, New York, NY 10021, USA. 12 4. School of Life Sciences, Tsinghua University, Beijing, 100084 China. 13 5. Department of Pharmaceutical & Biomedical Sciences, The University of Georgia, Athens, GA 14 30602, USA 15 6. Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, 16 ON K1H 8M5, Canada. 17 7. Department of Internal Medicine, Affiliated Cancer Hospital of Zhengzhou University & Henan 18 Cancer Hospital, Zhengzhou 450008, China 19 8. Department of Neurology, Comprehensive Cancer Center, The University of Alabama at 20 Birmingham, Birmingham, AL 35294, USA. 21 9. Division of Hematology and Oncology, University of Massachusetts Medical School, 364 22 Plantation St, Worcester, MA 01605, USA. 23 10. Cell Signaling Inc. 3 Trask lane, Danvers, MA 01923, USA. 24 11. Computational Biology Program, Sloan Kettering Institute, New York, NY 10021, USA. 25 12. Division of Preventive Medicine, The University of Alabama at Birmingham, Birmingham, AL 26 35294, USA. 27 28 * These authors contribute equally. 29 Corresponding Author: Xinyang Zhao Ph.D. email: [email protected], Tel: 205-975-5016 Fax: 205-975- 30 3335. 31 32 33 Keywords: PRMT1, RUNX1, c-MPL, GATA1, CNOT4, ubiquitylation, megakaryopoiesis, leukemia, 34 hematopoiesis, RNA metabolism, RNA splicing 35 36 Major Organism: Human 37 38 Impact Statement: Inhibiting PRMT1 enzymatic activity promotes megakaryocyte differentiation via 39 RBM15-mediated RNA metabolism, which is dysregulated in hematological malignancies. 40 1 41 Abstract: 42 RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We 43 demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue 44 R578 leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in 45 acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by 46 downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal 47 differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-mRNA 48 intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. 49 Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 50 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via 51 reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore 52 megakaryocyte differentiation for acute megakaryocytic leukemia. 2 53 Introduction: 54 RNA binding proteins control post-transcriptional processing such as alternative RNA splicing, 55 polyadenylation and protein translation, which is a prevalent part of gene regulation in normal cell 56 differentiation and in cancer development (Cabezas-Wallscheid et al., 2014; Chen et al., 2014; de Klerk 57 and t Hoen, 2015; Shapiro et al., 2011). Arginine methylation of RNA binding proteins by protein arginine 58 methyltransferases (PRMTs) regulates RNA splicing (Bedford and Clarke, 2009; Bezzi et al., 2013; 59 Cheng et al., 2007; Sinha et al., 2010), subcellular localizations (Matsumoto et al., 2012; Nichols et al., 60 2000; Tradewell et al., 2012) as well as the binding affinity to RNA molecules (Rho et al., 2007). 61 Nevertheless, the role of arginine methylation in regulating protein stability remains unknown. Here we 62 demonstrate that an RNA binding protein, RBM15, is methylated by PRMT1, which triggers its 63 ubiquitylation by an E3 ligase CNOT4. 64 65 RBM15 belongs to the Split Ends (Spen) family of proteins. Spen proteins are evolutionally conserved 66 RNA binding proteins, which are involved in transcriptional regulation of NOTCH, WNT and MAPK signal 67 transduction pathways (Chang et al., 2008; Chen and Rebay, 2000; Oswald et al., 2002; Rebay et al., 68 2000; Su et al., 2015). Recently SPEN and RBM15 have been shown to be essential for Xist-mediated X 69 chromosome inactivation (Chu et al., 2015; McHugh et al., 2015; Minajigi et al., 2015; Moindrot et al., 70 2015; Monfort et al., 2015). Genetic studies in Drosophila have shown that spen is required for cell-fate 71 decision during development (Kolodziej et al., 1995). FPA, the RBM15 homolog in Arabidopsis, controls 72 flowering via regulating alternative polyadenylation of antisense RNAs at the FLC locus (Hornyik et al., 73 2010). RBM15 is essential for the development of multiple tissues in mouse knockout models, in 74 particular, for the maintenance of the homeostasis of long-term hematopoietic stem cells and for 75 megakaryocyte (MK) and B cell differentiation (Niu et al., 2009; Raffel et al., 2009; Xiao et al., 2015). 76 Furthermore, RBM15 is involved in the chromosome translocation t(1;22) which produces the RBM15- 77 MKL1 fusion protein associated with acute megakaryoblastic leukemia (AMKL) (Ma et al., 2001; Mercher 78 et al., 2001). 3 79 80 Spen proteins consist of two domains: an RNA binding domain and a SPOC (Spen Paralog and Ortholog 81 C-terminal) domain. Previously, SPEN proteins such as RBM15 and SHARP have been shown to use 82 the SPOC domains to recruit histone deacetylases for transcriptional regulation of Notch pathway and 83 steroid receptor-dependent transcriptional regulation, and to recruit MLL complexes to promoters for 84 histone H3K4 methylation (Ariyoshi and Schwabe, 2003; Lee and Skalnik, 2012; Ma et al., 2007; Oswald 85 et al., 2002; Shi et al., 2001; Xiao et al., 2015). Additionally, RBM15 is also involved in RNA export 86 (Uranishi et al., 2009; Zolotukhin et al., 2008; Zolotukhin et al., 2009). RBM15 resides mainly within 87 nuclear RNA splicing speckles by confocal microscopy (Horiuchi et al., 2013b), suggesting that RBM15 is 88 involved in RNA splicing. However, how spen proteins control cell differentiation is not described at 89 molecular level. 90 91 In this report, we linked cellular differentiation to RBM15-regulated RNA metabolism using 92 megakaryocyte differentiation as a model. We demonstrated that RBM15 binds to specific introns of pre- 93 mRNA of genes such as RUNX1, GATA1 and TPOR (aka c-MPL or MPL), which play critical roles in 94 megakaryocyte differentiation, and to 3’UTRs of genes involved in RNA splicing and metabolic 95 regulation. Reducing RBM15 protein concentration by PRMT1-mediated methylation favors the 96 production of the alternatively spliced isoforms: RUNX1a, GATA1s and c-MPL-exon9-, a truncated c- 97 MPL isoform. We also found that RBM15 promotes megakaryocyte maturation in human primary cells. 98 Therefore, PRMT1-RBM15 pathway fine-tunes cell differentiation via controlling the RBM15 protein 99 concentration. 100 101 Results 102 103 RBM15 is methylated by PRMT1 104 4 105 In AMKL, MK differentiation is blocked. Gene expression data from AMKL patient samples (Bourquin et 106 al., 2006) shows a higher PRMT1 expression level than other types of acute myeloid leukemia. 107 Furthermore, high expression of PRMT1 is correlated with poor survival rate in acute myeloid leukemia 108 (Figure 1-figure supplement 1). These clinical data strongly support that PRMT1 might be a key player 109 in leukemogenesis. We applied bioorthogonal profiling of protein methylation (BPPM) technology (Figure 110 1-figure supplement 2) (Wang et al., 2011) to identify proteins methylated by PRMT1 in 111 megakaryocytes. We found that RBM15 is methylated at R578 by mass spectrometry analysis (Figure 1- 112 figure supplement 3A). Alignment of the RBM15 sequences covering the methylation site shows that 113 the methylation site is conserved across diverse species (Figure 1A) and downstream of the RBM15 114 RNA binding domains (Figure 1-figure supplement 3B). 115 116 We validated that two generic antibodies against mono-methyl arginine proteins and against di-methyl 117 arginine proteins (Cell Signaling Inc.), can recognize the methylated peptides from RBM15 (NP_073605) 118 R578 region specifically in dot blots (Figure 1-figure supplement 3C&D). We observed that affinity 119 purified Flag-tagged RBM15 protein is more efficiently methylated than the R578K mutant in western 120 blots in transfected 293T cells (Figure 1B, compare lanes 1 and 2) as well as in leukemia cells (Figure 121 1-figure supplement 3E). Given that the R578 is the last arginine in the RDRDRD repeat, we mutated 122 all three arginines, but we still detected low methylation background indicating other arginine residues 123 not in the repeat are methylated (Figure 1-figure supplement 3F). 124 125 To determine whether PRMT1 is responsible, we overexpressed the two major isoforms of PRMT1 (V1 126 i.e. Q99873 and V2 i.e. Q99873-3) respectively in 293T cells together